Ethanol exposure induces neurotoxicity that is related to neuroinflammation. Microglia serve as immune cells in the central nervous system (CNS) and have been implicated as the primary contributor to oxidative and nitrosative stress on neurons; however, little is known regarding the role of microglia in alcohol-induced neuronal dysfunction, especially in the context of neuron-microglia interplay. In this proposal, we will focus on elucidating the role of nitrosative stress in alcohol-induced microglial activation that ultimately leads to subsequent neuronal injury. We will employ novel mass spectrometric and proteomic methods to identify and quantify differential protein expression and changes in nitrosative stress-related protein modifications to test our working hypothesis that alcohol-induced neurotoxicity is mediated, at least in part, by nitrosative injury from microglial activation whichis in turn modulated by interaction with surrounding neurons. We propose two specific aims to determine the response of microglia to ethanol exposure and how that response is modified by interaction with neurons, and define the role of nitrosative protein modification in cellular dysfunction induced by ethanol exposure. In SA1, we will employ stable isotope labeling with amino acids in cell culture (SILAC) followed by quantitative mass spectrometric analysis to profile differential protein expression in these cell culture systems. In SA2, novel activity-based protein profiling (ABPP) probes will be utilized to accurately measure (through affinity purification followed by semi-quantitative mass spectrometry or fluorescence visualization) a pro-inflammatory protein marker, inducible nitric oxide synthase, in the cell culture models described in SA1. The results from our proposed studies will provide further insight into the role of microglia in ethanol-induced neurodegeneration and identify potential targets for new therapeutic strategies for the treatment or prevention of neuronal injury brought about by excessive and long-term alcohol consumption.